Methods for the production of non-covalently complexed and multivalent proteosome sub-unit vaccines

ABSTRACT

A continuous method for preparing proteosome-amphiphilic determinant vaccines for parenteral or mucosal administration using diafiltration or ultrafiltration technology. The amphiphilic determinants include lipopolysaccharides from gram negative bacteria, e.g.  S. flexneri, P. shigelloides  and  S. sonnei . Proteosomes are obtained from group B type 2b meningococci. The active proteosome-amphiphilic determinant complexes (non-covalent complexes) of the vaccine are formed using diafiltration or ultrafiltration to remove the detergent under non-static conditions. The use of diafiltration or ultrafiltration decreases processing time and the opportunity for contamination and further permits the use of ambient temperature and efficient scale-up. In addition, the process permits the reliable and continuous monitoring of the dializate which enhances the efficiency of the entire process. The time of dialysis for the production of a lot of vaccine is reduced from 7-10 days to less than 72 hours and usually less than 48 or 24 hours. The use of the process optimizes the presence of each antigenic component in the preparation of multivalent vaccines.

This application is a national stage application of PCT application No.PCT/US96/15002, filed Sep. 18, 1996, which is based on provisionalapplication No. 60/003,859, filed Sep. 18, 1995.

1. FIELD OF THE INVENTION

This invention concerns methods of production and compositions fornon-covalently complexed multivalent proteosome vaccines for mucosal andparenteral administration.

2. BACKGROUND OF THE INVENTION

In order for multivalent sub-unit vaccines to stimulate optimal immuneresponses to each of the components, the proper components should beappropriately associated and each be available to the immune system sothat they may be efficiently recognized and processed by cells of theimmune system. Prime examples of such non-covalently complexed vaccinesinclude proteosome vaccines which can consist of neisserial outermembrane protein proteosomes non-covalently complexed to a wide varietyof antigens including peptides, lipopeptides, transmembrane or toxoidedproteins, polysaccharides or lipopolysaccharides (LPS) (patentapplication Ser. No. 07/065,440 filed Jun. 23, 1987 “Immunogenic peptidevaccines and methods of preparation”; Ser. No. 07/336,952 filed Apr. 12,1989 Immunopotentiaing system for large proteins and polypeptides”; Ser.No. 07-958,426 filed Oct. 8, 1992 “Oral or Intranasal Vaccines UsingHydrophobic Complexes Having Proteosomes and Lipopolysaccharides”; Ser.No. 08/029,666 filed Mar. 11, 1993 “Immunopotentiating Systems forPreparation of Immunogenic Materials”; Ser. No. 08/143,365 filed Oct.29, 1993 “Immunopotentiating Systems for Preparation of ImmunogenicMaterials”; Ser. No. 93/10,402 filed Oct. 29, 1993 “Submicron Emulsionsas Vaccine Adjuvants”; Ser. No. 08/063,613 filed May 18, 1994 Solid FatNanoemulsions as Vehicles for Vaccine Delivery” and publications Orr,N., Robin, G., Cohen, D., Arnon, R. and Lowell, G. H. (1993).Immunogenicity and Efficacy of Oral or Intranasal Shigella flexneri 2aand Shigella sonnei Proteosome-Lipopolysaccharide Vaccines in AnimalModels. Infect. Immun. 61:2390; Mallett, C. P., T. L. Hale, R. Kaminski,T. Larsen, N. Orr, D. Cohen, and G. H. Lowell. 1995. Intranasal orintragastric immunization with proteosome-Shigella lipopolysaccharidevaccines protect against lethal pneumonia in a murine model ofshigellosis. Infect. Immun. 63:2382-2386.; Lowell G H, Kaminski R W,Grate S et al. (1996) Intranasal and intramuscularproteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines:immunogenicity and efficacy against lethal SEB intoxication in mice.Infec. Immun. 64:1706-1713.; Lowell, G. H. (1990) Proteosomes,Hydrophobic Anchors, Iscoms and Liposomes for Improved Presentation ofPeptide and Protein Vaccines. in New Generation Vaccines: G. C. Woodrowand M. M. Levine, eds. (Marcel Dekker, NY). Chapter 12 (pp. 141-160) andLowell, G. H., W. R. Ballou, L. F. Smith, R. A. Wirtz, W. D. Zollingerand W. T. Hockmeyer. 1988. Proteosome-lipopeptide vaccines: enhancementof immunogenicity for malaria CS peptides. Science 240:800.)

The contents of all the documents cited herein are expresslyincorporated by reference.

For practical application in administering vaccines to protect againstdisease, it is frequently necessary to deliver several such antigens atthe same time usually due to the fact that individuals are susceptibleto the contraction of diseases caused by a variety of organisms.Moreover, several organisms, whether or not they are related to oneanother, often are endemic in the same location and thereforeindividuals requiring protection may need vaccination with several typesof vaccines.

In the past, the production of vaccines that require non-covalentcomplexing of components has been accomplished using simple dialysis inwhich components are placed in dialysis tubing in the presence ofdialyzable detergent and the mixture is dialyzed for 7-10 days toattempt to remove the detergent. The practical disadvantages of thissystem tend to severely preclude the advanced development andcommercialization of this technology for several reasons including 1)Time: Length of time of the procedure: The need to use GMP resources forweeks while the vaccine is dialyzing is impractical both due to theexcess costs involved and the increased opportunity for breakdown orcontamination of mechanical or biological components during thisextended period of time; 2) Contamination: Increased opportunity forcontamination: dialysis tubing is difficult to sterilize, dialysistubing requires manually opening and closing the system thereby exposingthe components to contamination during both the loading and unloadingprocess. Since many days transpire between loading and unloading thetubing, the risk of a small contamination in the initial days of theprocess may readily be magnified during the many days of dialysis torender this method useless for practical vaccine manufacture. The riskof puncturing the bag can result in loss of product. 3) Temperature:Necessity to perform the dialysis at 40° C. due to the extensive timeinvolved; 4) Volume of dialyzing fluids: In order to manufacture vaccinefor scale-up of the process, the use of massive amounts of dialysisfluid would be necessary since a 200:1 ratio of liquid outside to thedialysis tubing to inside the tubing is typically required. Therefore,for example, the production of a pilot lot of two liters of vaccinewould require 400 liters of fluid outside the tubing per day—4,000liters per 10 days—and the production of a production lot of 20-200liters would require 40,000-4,000,000 liters. These amounts are wastefuland impractical compared to the method used in the instant invention;Dialysis tubing is not scalable since large amounts of product isproblematic and 5) Inability to readily measure completeness of removalof the detergent so as to maximize vaccine effectiveness. Since thedialysis bag is placed in a container with 200 volumes of buffer, theongoing measurement of detergent removal is neither practical norfeasible and 6) In addition, no method has been described for themeasurement of the presence of the detergent used in the preferredembodiment, Empigen BB.

The second problem solved in this invention is the demonstration of themethod of producing and delivering multivalent vaccines. Components caneither be made together or produced separately and mixed together priorto administration. The instant invention demonstrates the optimal way ofpreparing such multivalent vaccines.

3. SUMMARY OF THE INVENTION

The subject of the instant invention broadly relates to the productionand manufacture of proteosome-amphiphilic determinant vaccines designedfor either parenteral or especially for mucosal administration including, but not limited to, respiratory (e.g. including intranasal,intrapharyngeaeal and intrapulmonary), gastro-intestinal (e.g. includingoral or rectal) or topical (e.g. conjunctival or otic) administration toinduce both systemic (serum) and mucosal (including respiratory andintestinal) antibody responses. An amphiphilic determinant is a moleculehaving hydrophobic and hydrophilic regions which, when appropriatelyformulated with proteosomes, align with the proteosomes to for a complexwhich elicits an immunologic response in a subject. Typical amphiphilicdeterminants include glycolipids, liposaccharides (including detoxifiedlipopolysaccharides), lipopeptides, transmembrane, envelope or toxoidedproteins, or proteins or peptides with intrinsic hydrophobic amino acidanchors. These determinant materials can be obtained from gram negativebacteria including eschefichia, klebsiella, pseudomonas, hemophilusbrucella, shigella and neisseria. More specifically, the inventionrelates to proteosome vaccines in which meningococcal outer membraneprotein proteosome preparations (prepared from any strain of N.meningiditis or N. gonorrhea or other neisserial species) arenon-covalently complexed to native or detoxified shigella or neisseriallipopolysaccharides or lipooligosaccharides to form vaccines designed toprotect against diseases caused by gram negative organisms that containany of the component parts of the complex including meningococci orshigellae. More specifically, the invention relates to proteosomevaccines that contain LPS that induce antibody responses that recognizetype-specific somatic polysaccharide O-antigens of shigellalipopolysaccharides and thereby confer homologous protection againstshigellosis. Still more specifically, the lipopolysaccharides that, whencomplexed to proteosomes induce such anti-shigella protective immuneresponses are prepared and purified from either Shigella sonnei orPlesiomonas shigelloides for immunity against Shigella sonnei disease,from Shigella flexneri 2a for immunity to Shigella flexneri 2a disease,and so forth, using LPS derived from homologous or antigenicallycross-reacting organisms to confer homologous immunity againstshigellosis caused by S. flexneri 2a (or 3a etc.), S. boydii, S. sonneietc. Still more specifically, the instant invention describes thesuccessful administration of proteosome-shigella vaccines that aremultivalent in that two independently made proteosome vaccines usingshigella LPSs derived from S. flexneri 2a (for S. flexneri 2a disease)and from P. shigelloides or S. sonnei (for S. sonnei disease) areadministered together thereby inducing antibodies that recognize the twoorganisms and thereby conferring protection against the two types ofdiseases. Most specifically, the instant invention relates to aproteosome-shigella LPS vaccine in which proteosomes from group B type2b meningococci are complexed to P. shigelloides LPS using hollow fiberdiafiltration technology to produce a vaccine administered by mucosalrespiratory and/or gastro-intestinal routes to induce antibodies thatrecognize the somatic O-antigen LPS of S. sonnei and thereby protectagainst shigellosis caused by this organism. Other conventionalultrafiltration/diafiltration are envisioned, e.g. platform membrane andmembrane cartridge.

The present invention provides methodology to produce non-covalentlycomplexed vaccines in a manner that 1) Decreases the time required, 2)Decreases the opportunity for contamination, 3) Increases thetemperature to ambient temperature that such vaccines can be produced,4) Allows for efficient scale-up of the production process so as torequire minimum use of reagents, 5) Allows for reliable and efficientsampling of dialysate so as to be able to repeatedly measure rate ofremoval of the detergent so as to optimize efficiency of the operation.This leads directly to an increase in the complexing efficiency ofvaccine so as to produce vaccine with measurably greater immunogenicityat lower doses. In this manner, the overall quality of the product issignificantly enhanced. 6) In addition, a method is described to measurethe presence of the detergent used in the preferred embodiment, EmpigenBB. Other dialyzable detergents can be used in place of Empigen BB.Furthermore, using the method of the instant invention, it has beendemonstrated that the vaccine can be lyophilized and re-hydrated in sucha manner as to retain optimal vaccine potency as measured by vaccineimmunogenicity.

The method of the instant invention entails the use of a hollow fiberultrafiltration/diafiltration cartridge to effect complexing by removalof the detergent. By varying the size of the housing of the cartridge,the time of dialysis for production of a lot of vaccine can be reducedfrom >7-10 days to less than 72 hours and usually less than 48 or 24hours. Since this short time is used, the reaction temperature can beincreased from 4° to normal room temperature without compromising theprocess or the integrity of the products. While the process wasperformed at about 20° C., temperatures between 0° and 40° C. arecontemplated. Since the system is closed, there is a highly reducedpotential for contamination. In addition, by increasing the size of thehousing, an exceedingly large amount of material can be processed in arelatively short period of time thereby allowing for efficient andreproducible scale-up of the procedure for commercial development.Furthermore, the permeate can be repeatedly sampled to measure theremoval of the detergent as can be accomplished using the test of theinstant invention to measure the presence of the detergent empigen BB,the detergent used in the preferred embodiment. The uniqueness of thismethodology needed experimental verification since it was not obviousthat the complexing and the structure of the vaccine into immunogenicmaterials that was accomplished by slowly dialyzing over 7-10 days usinga stationary dialysis bag could be equalled or improved upon using thehollow fiber technology in which the moieties to be complexed are movingthrough tubes at exceedingly high flow rates compared to that of thestationary dialysis tube.

Since the nominal molecular weight cutoff of the membrane used can beselected to be from 1,000, 3,000, 5,000, 10,000, 30,000, 50,000 orgreater, depending on the size of the components to be non-covalentlycomplexed, this system is readily adaptable to the complexing of nativeor detoxified lipopolysaccharides, lipids, peptides, lipopeptides,liposaccharides, polysaccharides, gangliosides or transmembrane,envelope or native or toxoided proteins to each other or tomeningococcal outer membrane protein preparations of proteosomes.

The resultant product can be used as a vaccine administered eitherparenterally or mucosally i.e. via the respiratory or gastro-intestinaltract e.g. intranasally, orally, by oropharyngeal inhalant, topically orrectally. In the example given, it is shown that proteosomes arenon-covalently complexed to P. shigelloides LPS to form a vaccine thatinduces the anti-S. sonnei LPS responses necessary for protectionagainst S. sonnei shigellosis.

The methodology has three stages: a preliminary operation; complexingthe proteosome with an amphiphilic determinant e.g. P. Shigelloides LPS;followed by a sterile filtration.

To optimally deliver multivalent vaccines, the present invention showsthat the best method is to make the specific vaccines individually andthen immunize with the two individually made vaccines, each at theoptimal concentration, on the same day. Other possibilities such asmaking hybrid vaccines during the formation of the non-covalentcomplexes have also been accomplished or are envisioned.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Intranasal immunization with proteosome-P. shigelloides LPSVaccine: serum IgG responses elicited by several preparations atdecreased doses.

FIG. 2: Intranasal immunization with Proteosome-P. shigelloides LPSvaccine: serum IgA responses elicited by several preparations atdecreased doses.

FIG. 3: Administration of the proteosome-P. shigelloides LPS (for S.sonnei shigellosis) and the proteosome-S. flexneri 2a vaccines togetheron the same day or weeks apart results in good immunogenicity to boththe S. sonnei and S. flexneri 2a LPS components.

FIG. 4: Immunogold labelled electron micrograph of proteosome-shigellaLPS vaccine showing association of shigella LPS with proteosomevesicles.

FIG. 5: Proteosome-P. Shigelloides LPS vaccine protects against lethalShigella sonnei pneumonia in an animal model of Shigellosis.

FIGS. 6A-D: Intranasal or oral immunization with proteosome-Shigellaflexneri 2a LPS vaccines induce anti-shigella LPS IgG and IgA in serumand IgA in intestinal and lung lavage fluids that can last 30-60 dayspost-immunization.

FIG. 7: Intranasal or oral immunization of humans with one or two dosesproteosome-p. shigelloides LPS vaccines for Shigella sonnei usingdifferent vaccine amounts: Induction of anti-shigella LPS IgA, IgG andIgM peripheral blood ASC responses, and serum, salivary and urinaryantibody responses.

FIG. 8: LPS in HPLC fractions after applying uncomplexed P. shigelloidesLPS and Demonstration of non-covalent complexing of LPS and proteosomeproteins by co-elution of LPS and proteins in HPLC fractions afterapplying proteosome-P. shigelloides LPS vaccine.

FIGS. 9A-B: Bactericidal antibody response of mice to meningococcalouter membrane protein-detoxified lipooligosaccharide vaccine, geometricmean reciprocal titers against meningococcal strain 9162

5. DETAILED DESCRIPTION OF THE INVENTION Example 1

This example is detailed for production of proteosome-P. shigelloidesvaccine containing approximately 2-3 grams of protein. The procedure isequally applicable for scale-up using 10-1000 fold more material withappropriate scale-up of housing size of the membrane cartridge andappropriate increases in the volumes. Using the appropriate sizemembranes with appropriate size molecular weight cutoffs to retain theantigens to be complexed, this procedure is equally applicable forcomplexing of proteosomes to other native or detoxifiedlipopolysaccharides, lipids, peptides, lipopeptides, liposaccharides,polysaccharides, gangliosides or transmembrane, envelope or native ortoxoided proteins to each other or to meningococcal outer membraneprotein preparations of proteosomes. Empigen BB is the detergent used asan example in this procedure; the procedure applies equally to anydialyzable detergent that solubilizes the components. This procedureuses A/G technology cartridges but is equally applicable to any brandcartridge type ultrafiltration/diafiltration system.

5.0.0.1 Preliminary Operation

5.0.0.1. Sterile filter a stock solution of 30% Empigen BB, which is thedetergent used in this process.

5.0.0.2. Prepare 2×TEEN/2% Empigen (0.1M Tris, 0.02 M disodium EDTA, 0.3M sodium chloride, WFI and 2% Empigen, pH 8.0)

5.0.0.3. Prepare 150 L of a TNS (Tris Normal Saline) solution containing0.05M Tris, 0.15M sodium chloride pH 8.0 for the purpose of dialyzingout the detergent.

5.0.0.4. Prepare 10 liters of Sodium Hydroxide for UF cartridgecleaning.

5.0.0.5. Thaw the required amount of Shigella flexneri 2aLipopolysaccharide (LPS).

5.0.1. Complexing Proteosomes with S. flexneri 2a LPS

5.0.1.6. Add an equal volume of 2×TEEN buffer to the volume of LPS andmix well.

5.0.1.7. Thaw and measure an amount of bulk proteosomes, in mg's, equalto the amount of LPS thawed in step 5.

5.0.1.8. To the proteosomes, add 30 ml of sterile filtered empigen perliter. Mix well and combine with the LPS from step 5.

5.0.1.9. Mix both components well. Usually 15 minutes on a stir plateand stir bar combination is adequate.

5.0.1.10. To dialyze the detergent out so that proteosome complexescould form, set up an ultrafiltration system (based on tangential flowtechnology} using a hollow fiber cartridge with a nominal molecularweight cutoff (NMWC) pore size of 10,000 manufactured by A/G Technology,Inc. connected to sterilized silicone tubing and a peristaltic pump.Sanitary pressure gauges are placed on the inlet and outlet side of thecartridge to monitor pressure throughout the run. A back pressure valveis placed on the outlet side of the cartridge to as to adjust backpressure.

5.0.1.11. The ultrafiltration system is cleaned and sanitized with WFIand 0.5N NaOH at a temperature of 50° C. for a time greater than 60minutes. This system is flushed with WFI and equilibrated with TNSbuffer prior to use.

5.0.1.12. A sterile reservoir vessel is placed in line so that the inletliquid is pulled from the vessel and the return retentate returned tothe same reservoir. As liquid passes through the cartridge membrane(permeate or filtrate) it is replenished by TNS buffer either by directaddition or by a continuous feed system. Set up a continuous feed systemby connecting silicone tubing to a vessel containing TNS buffer to thesample reservoir that is then air tight. As the liquid is removed byfiltration from the sample reservoir a vacuum is created causing “TNS”buffer to be pulled from the its vessel into the sample reservoir. Ifthe system remains air tight the sample level it the reservoir remainsconstant.

5.0.1.13. Start the recirculation pump to begin diafiltration. Adjustthe pump speed and the back pressure valve to obtain a back pressure of15±4 psi.

5.0.1.14. Verify progressive removal of Empigen BB by testing permeatesamples taken every 10-15 liters using the Empigen Precipitin Test. Thistest consists of making serial dilutions of the permeate and of astandard solution containing a known quantity of empigen BB, adding HClto acidify the solutions and then adding serial dilutions of SDS (sodiumdodecyl sulfate) to form a checkerboard-type assay. Maximum precipitatewill form when equivalent amounts of empigen and SDS are present. Inthis manner, the amount of empigen present can be quantitated by notingthe dilution of the SDS, the permeates and of the standards at which themaximum precipitate is formed. As the diafiltration progresses, theamount of empigen becomes less and the tube containing less SDS will bethe tube with the maximum precipitate. The presence of precipitate isquantitated by measuring the OD at 600 nm using a spectrophotometer.

5.0.1.15. For the amount of components used in this example, continuediafiltration until at least 120 liters of permeate has been processedand there is a lack of significant precipitate in the Empigen PrecipitinTest (Maximum OD 600 nm is less than 0.05).

5.0.1.16. After dialysis is complete concentrate the product to a finalvolume to get an OD₂₈₀ value close to 6.0. Drain the system and washwith 300-400 ml of “TNS” buffer. Drain the system again and combine bothpools. Clean the cartridge by recirculating large amounts of WFIfollowed by 0.5N NaOH at 50° C. for >60 minutes. Rinse out the NaOH withWFI and store the system in 0.01N NaOH.

5.0.2. Sterile Filtration

5.0.2.17. The complexes may, if desired, be sterile filtered. Proteosomeconcentration can be adjusted before or after 0.22μ filtering. Sterilefiltration is normally performed with filter units from Millipore Corp.These units are called Millipaks and they come in various sizes. Thefiltered bulk is stored at 4° C. until the fill operation and is testedfor sterility.

5.0.3.0 Specific Purpose

Combine bulk GMP preparations of meningococcal outer membrane proteinproteosomes and S. flexneri 2a lipopolysaccharide into non-covalentcomplexes by removal of detergent.

5.0.3.1 Applications

This GMP procedure results in the production of a bulk preparationformation of non-covalent complexes of proteosomes with S. flexneri 2alipopolysaccharide for use as a vaccine against S. flexneri 2ainfection.

Example 2

Noncovalent complexing of bulk Neisseria meningitidis strain 9162purified outer membrane proteins (proteosomes) and alkaline detoxifiedN. meningitidis L8 lipooligosaccharide purified from strain 8532.

5.1.0 Bulk 9162 outer membrane protein (proteosomes), lot 0136 and bulkdetoxified meningococcal L8 Lipooligosaccharide (LOS) lot 0203 weretaken from storage and thawed at room temperature. A volume (182 ml) ofthe bulk LOS containing 500 mg LOS in sterile distilled water wascombined with a volume (306 ml) of bulk proteosomes containing 400 mg ofprotein in buffer containing 0.05 M Tris-HCl, 0.15 M NaCl, 0.01 M EDTA,and 0.1% Empigen BB.

5.1.2 Empigen BB (30% solution) was sterile filtered through a 0.22 μmpore size filter and {fraction (1/60)}^(th) volume added to the combinedproteosomes, LOS solution and stirred at room temperature for 1 hour.

5.1.3 The detergent buffer solution was removed from the proteosomes,LOS solution and replaced with sterile distilled water byultrafiltration using an A/G Technology ultrafiltration cartridgeUFP-3-C-6 with a 3000 molecular weight cut off pore size.

5.1.4 The procedure for set up, sanitization, washing, washing, andcleaning of the apparatus was the same as described in example 1. Inletpressure was 15±4 PSI and the permeate flow rate was 175 ml per minute.

5.1.5 The permeate was tested after each 3 to 4 liters for the presenceof Empigen BB detergent by the precipitation method described inexample 1. The ultrafiltration was continued until no precipitate wasobtained in the test plus an additional 5 liters. A total of 37 litersof permeate was collected.

5.1.6 The retentate was clear and was sterile filtered through a 0.22 μmpore size filter assayed for protein and LOS and stored as a bulkproduct at 4° C.

5.1.7 The bulk product was diluted to a concentration of 0.2 mg ofprotein per ml and the concentration of NaCl adjusted to 0.15 M.Thimerasol at 0.01% was added as a preservative, and the resulting bulkwas dispensed under sterile conditions into final container vials,labelled and stored at −70° C.

5.1.8 This product was tested in mice for immunogenicity when given as asaline solution and adsorbed to aluminum hydroxide gel as adjuvant. Theresults are given in the Table 1 below and show that the vaccinecomplexes were immunogenic when given i.p. in mice.

TABLE 1 Bactericidal antibody response of mice to meningococcal outermembrane protein-detoxified lipooligosaccharide vaccine, geometric meanreciprocal titers against meningococcal strain 9162 Vaccine DoseAdjuvant Day 0 Day 28 Day 42 proteosomes- 1 μg None <8 <8  16 dLOS 3 μgNone <8 <8 128 Lot 0271 10 μg  None <8 <8 512 protesomes- 1 μg Al(OH)₃<8 <8 512 dLOS 3 μg Al(OH)₃ <8 <8 512 Lot 0271 10 μg  Al(OH)₃ <8 642048  Note: Vaccine was given intraperitoneally at 0 and 28 days togroups of ten outbred CD-1 mice.

5.1.9 The complexing of the proteosomes and LOS was verified by columnchromatography of the proteosomes and LOS components before and aftercomplexing (FIG. 9). Chromatographic analysis of bulk meningococcal L8LOS and the final proteosomes/LOS complexes on Sephacryl S300 lowpressure column. The column was run in 0.05 M Tris-HCl, 0.01 M EDTA,0.15 M NaCl buffer. The assay for protein was by absorbance at 280 nm,and the assay for LOS was by inhibition of an ELISA (enzyme linkedimmunosorbant assay) in which an L8 monoclonal antibody binding topurified L8 LOS adsorbed to the plastic plate was inhibited by serialdilutions of the fractions off the Sephacryl column. Before complexing,the LOS came off the column in a broad peak centered at fraction 39.After complexing, the LOS co-eluted with the proteosomes in a peakapproximately centered at fraction 33.

5.1.10 Specific Purpose

Combine bulk GMP preparations of meningococcal outer membrane proteinproteosomes and detoxified meningococcal lipopolysaccharide intonon-covalent complexes by removal of detergent.

5.1.11 Applications

This GMP procedure results in the production of a bulk preparationformation of non-covalent complexes of proteosomes with detoxifiedmeningococcal lipopolysaccharide for use as a vaccine against N.meningitides (meningococcal) infection.

Example 3

This example is also directed to the production of proteosome-P.shigelloides vaccine. The procedure is equally applicable for scale-upusing 10-1000 fold more material with appropriate scale-up of housingsize of the membrane cartridge and appropriate increases in the volumes.

5.8.1 Preparation of 25 mL Sterile-Filtered Empigen BB (30% Solution)

5.8.1.1 Use a 100 ml Nalgene disposable filter unit with 0.2μ pore sizemembrane filter to sterile filter about 25 ml of Empigen BB (30%solution).

5.8.2 Prepare 4 L of TEEN 2× with 2% Empigen BB (consisting of 0.1 MTris, 0.02 M disodium EDTA, 0.3 M sodium chloride, WFI and 2% solutionof empigen BB).

5.8.3 Prepare a 10 L solution of “TNS” fifteen times to total 150liters: TNS consists of 0.15 M sodium chloride, 0.05 M Tris Buffer pH8.0±0.2

5.8.4 Prepare 10 liters of 0.5 N Sodium Hydroxide

5.8.6 If necessary, thaw bulk P. shigelloides lipopolysaccharide (LPS)to prepare for complexing with proteosomes and record the lot number,and concentration of the LPS, the required amount of LPS in mg and thecalculated volume of LPS to remove. ps 5.9.0 Complexing Proteosomes withP. shigelloides LPS

5.9.1 Preparation of LPS for Complexing

5.9.1.2 Pool the aliquots of bulk LPS into a clean, sterile, calibrated5 L bottle. Measure the total volume and determine the total amount ofLPS.

5.9.1.3 Add a volume of 2×TEEN buffer equal to the volume of LPS used instep

5.9.1.2, measure combined total volume, and then add a stir bar and mixwith stir bar and stir plate combination for 15±2 minutes.

5.9.2 Preparation of Proteosomes for Complexing

5.9.2.1 Record the total time, if any, proteosomes was allowed to thaw.

5.9.2.2 Pool the aliquots of bulk proteosomes into a clean, sterile, 1 Lgraduated cylinder. Measure the total volume and determine the totalamount of proteosomes.

5.9.2.3 From the cylinder in step 5.9.2.2, transfer to another clean,sterile, 1 L graduated cylinder, a volume of proteosomes containing theamount in mg equal to the mg amount of LPS used in step 5.9.1.2.

5.9.2.4 To the cylinder containing the proteosomes, add 0.22μ filtered30% empigen BB using 30 ml empigen per L proteosomes. Mix with a stirbar for 15±2 minutes.

5.9.3 Combine Protesomes with the P. Shigelloides LPS

5.9.3.1 Under gentle stirring with a stir bar, add the proteosomes fromstep

5.9.3.2 to the graduated 5 L bottle with the LPS and stir for 15±2minutes.

5.9.3.3 Remove four 1 ml samples and store at −75° C. for latermeasurement of the concentration of protein by the Lowry method and LPSby KDO assay.

5.9.4 Removal of Detergent by Ultrafiltration/Diafiltration

5.9.4.1 Ultrafiltration System Details System: A/G Technology, Inc.hollow fiber cartridge, 10,000 NMWC pore size, housing size 6,ultrafiltration cartridge Note:It is necessary to condition newcartridges to flush out glycerol. This can be done by flushing 6 litersof WFI through the 6 sq/ft cartridges or less with no back pressure. Thepermeate solution should not be recycled to the feed reservoir. Note:Allultrafiltration steps will be follow by cleaning, sanitization, andstorage steps. New cartridges have to be cleaned and sanitized prior touse.

5.9.4.2 Set-up and sanitize the A/G ultrafiltration system. Clean andsanitize the system by recirculating for a minimum of 60 minutes with2-3 liters of 0.5 N Sodium Hydroxide at an initial temperature of 50±5°C. Flush the system with 4-5 liters of WFI followed by recirculating 2-3liters of “TNS” (0.05 M Tris/normal saline buffer pH 8.0±0.2, for atleast 20±5 minutes.

5.9.4.3 Measure the hold up volume in the UF system (including tubing)by transferring inlet and outlet lines filled with liquid out of thereservoir and into a 1 liter graduated cylinder, then pump until the UFsystem is empty.

5.9.4.4 Place a 2 liter side-armed vessel in a pan and pack the vesselwith wet ice for the duration of the diafiltration procedure.

5.9.4.5 Transfer 1.7-1.9 L of the bulk proteosome-LPS mixture from step5.9.3.3 into the 2 L side-armed vessel. Place a two-holed stopper fittedwith two 10 ml pipettes on the vessel and connect the inlet and outlettubing from the diafiltration system to the vessel pipettes.

5.9.4.6 Turn on the recirculation pump and adjust the pump setting to4-6. Adjust the back pressure clamp to obtain an inlet pressure of 15±4psi. Concentrate to a total volume of 1.0±0.1 L including the hold-upvolume

5.9.4.7 Transfer 1±0.1 L of mixture from step 5.9.3.3 to the 2 Lside-armed vessel and repeat step 5.9.4.6.

5.9.4.8 Continue transferring as in step 5.9.4.7 and concentrating as instep 5.9.4.9 until all the proteosome-LPS mixture from step 5.9.3.3 hasbeen transferred to the 2 L side-armed vessel and the retentate volumeis 1.4±0.2 L which includes the hold-up volume

5.9.4.10 Set up the ultrafiltration apparatus for continuous feed of TNS(0.05 M Tris, normal saline) into the 2 liter side-armed vessel asliquid is removed through the membrane. Set up a reservoir using a 10liter bottle containing TNS and fitted with a tube extending to thebottom of the bottle. Connect the inlet and outlet lines from theultrafiltration system. Turn on the recirculation pump and adjust thepump setting to 4-6. Adjust the back pressure clamp to obtain an inletpressure of 15±4 psi.

5.9.4.11 Measure the permeate flow rate on the UF unit and record theinlet pressure when the measurement is taken.

5.9.4.12 Collect a 15 to 20 ml sample of the permeate every 12±0.5liters processed, apply an in-house label (“Permeate No. P1, P2, P3etc.”) and record the time, volume processed and maximum O.D.₆₀₀ of thesample. Verify progressive removal of empigen by testing the samples forthe presence of Empigen BB using the Empigen Precipitin Test. This testconsists of making serial dilutions of the permeate, and, separately, ofa solution containing a known quantity of empigen BB, adding HCl toacidify the solutions and then adding serial dilutions of SDS (sodiumdodecyl sulfate) to form a checkerboard-type assay. Maximum precipitatewill form when equivalent amounts of empigen and SDS are present. Inthis manner, the amount of empigen present can be quantitated by notingthe dilutions of the permeates and the SDS in which the precipitate isformed and comparing those dilutions to those of the standards. As thediafiltration progresses, the amount of empigen becomes less and thetube containing less SDS will be the tube with the maximum precipitate.The presence of precipitate is quantitated by measuring the OD at 600 nmusing a spectrophotometer.

For the amounts of components used in this example, continuediafiltration until at least 120 liters of permeate has been processedand there is a lack of significant precipitate in the Empigen precipitintest (Maximum OD 600 nm less than 0.05).

5.9.4.13 Concentrate the product to a final retentate volume of 500±50ml including the hold-up volume (step 9.4.3). Remove a 0.5 ml sample,dilute the sample 1:10, and measure the O.D.₂₈₀.

5.9.4.14 The desired minimum O.D. of the concentrated retentate is 5.7.If the O.D. is less than 5.7, continue concentrating to 400±50 mlincluding the hold-up volume (step 5.9.4.3) and repeat the O.D.₂₈₀.

5.9.4.15 With the retentate lines out of the retentate solution and thefiltrate outlet lines closed, slowly pump in the reverse direction untilthe cartridge and lines are empty. Transfer the feed line and retentatelines from the retentate bottle to a new clean vessel with 350±50 mlTNS. Reverse the pump and slowly recirculate the TNS for 2-3 minutes.

5.9.4.16 After recirculating the TNS, stop the pump, drain the system,collect the retentate wash solution into a clean, sterile, 1 L graduatedcylinder and measure the volume and the O.D.₂₈₀.

5.9.4.17 Combine the original retentate solution (step 5.9.4.12 or5.9.4.13) with the wash retentate solution (step 5.9.4.15) and measurethe final volume of retentate. Store the retentate bulk at 4° C.±2° C.until aseptic filtration.

5.9.4.18 Clean the filtration unit using WFI, followed by 0.5 N sodiumhydroxide at an initial 50±5° C. for a minimum of 60 minutes. Rinse thecleaning agent out with of WFI then flush with 3-4 liters of 0.1 N NaOHas the storage agent.

5.9.5 Sterile Filtration

The complexes may, if desired, be sterile filtered. Obtain the bulkcomplexed proteosomes and LPS and inspect for the presence of anyprecipitate or cloudiness. If the bulk complexed product is clear,aseptically filter it using a Millipak 40, 0.22μ filtration unit into asterile, depyrogenated vessel. This step is to be done in a SterileCabinet.

5.9.6 Specific Purpose

Combine bulk GMP preparations of meningococcal outer membrane proteinproteosomes and P. shigelloides lipopolysaccharide (for S. sonnei) intonon-covalent complexes by removal of detergent.

5.9.7 Applications

This GMP procedure results in the production of a bulk preparationformation of non-covalent complexes of proteosomes with P. shigelloides2a lipopolysaccharide for use as a vaccine against Shigella sonneiinfection.

5.10 Immunogenicity Studies

5.10.1 Immunogenicity of Proteosome-P. ShigelloideS LPS Vaccine UsingHollow Fiber Ultrafiltration/Diailtration

FIGS. 1 and 2 demonstrate that three different proteosome-shigella LPSvaccine preparations made using the hollow fiber (HF) diafiltrationtechnique (HF-1, HF-2 and GMP/HF-3) to remove detergent and facilitatecomplexing are more effective at the lowest, most stringent dose tested,than preparations made using simple dialysis tubing (DT). In otherwords, stronger IgG (FIG. 1) and IgA (FIG. 2) immune responses wereelicited by vaccines produced using the instant invention than by theolder, less efficient technology. Since stronger immune responses resultin better levels of protection, these data clearly show that the instantinvention results in vaccines that are not only more efficient to makebut also more powerful. Furthermore, the stronger responses are elicitedby the vaccine when administered at the lowest dose (0.1 ug per dose)thereby indicating that less vaccine would be needed using the instantinvention than would be required using the older technology. Since inthe formulation of multivalent vaccines requires many differentantigens, in order to minimize the total amount of vaccine administered,it is highly advantageous to be able to use vaccine components that areeffective at lower doses. Thus, the technology of the instant inventionto produce vaccines using the hollow fiber technology directly impactsand facilitates the ability to formulate multivalent vaccines having awide range of specificities. Since it is advantageous for commercialdevelopment of vaccines to be able to lyophilize vaccines, we weregratified to discover that lyophilization of the HF vaccine complexesfrom a water solution, surprisingly resulted in vaccine thatconsistently elicited responses that were among the highest at all dosestested. Thus, higher immunogenicity for anti-LPS IgG FIG. 1) and IgA(FIG. 2) was found when 0.1 ug of vaccine made by the HF technique (withor without lyophilization) was administered. These data are significantsince the scale-up and GMP procedure of the instant invention used theHF technology as indicated in FIGS. 1 and 2 by the cross-hatched barrepresenting GMP/HF-3.

5.10.2 Immunogenicity Studies Using Multivalent Vaccine

As shown in FIG. 3, Administration of the proteosome-P. shigelloides LPS(for S. sonnei shigellosis) and the proteosome-S. flexneri 2a vaccineson the same day or weeks apart results in good immunogenicity to bothLPS components: S. sonnei LPS and S. flexneri 2a LPS. Scheduled 1consisted of five mice intranasally immunized on days 0 and 21 with theproteosome- S. sonnei LPS vaccine (10 micrograms each of proteosomes andLPS). Mice were bled four weeks after the last immunization. Scheduled 2consisted of five mice intranasally immunized on days 0 and 21 with theproteosomes-S. flexneri 2a LPS vaccine (10 micrograms each ofproteosomes and LPS). Mice were bled four weeks after the lastimmunization. Schedule 3 consisted of five mice intranasally immunizedon days 0 and 21 with the proteosomes-S. flexneri 2a LPS vaccine (10micrograms each of proteosomes and LPS). Mice were bled four weeks afterthe last immunization. Schedule 4 consisted of four mice intranasallyimmunized on days 0 and 21 with the proteosome- S. sonnei LPS vaccine(10 micrograms each of proteosomes and LPS) and then on days 49 and 70with the S. flexneri 2a LPS vaccine (10 micrograms each of proteosomesand LPS). Mice were bled four weeks after the last S. flexneri 2aimmunization.

5. 11 Electron Micrograph of Proteosome-Shigella Vaccine

The association of shigella LPS with proteosomes is clearly anddramatically depicted in the electron micrograph shown in FIG. 4. Inthis Figure, the proteosome vesicles are studded with radio-opaque blackdots. These dots are god beads linked to secondary antibodies thatrecognize specific anti-shigella LPS antibodies. Thus, the presence ofgold dots indicates the presence of shigella LPS. Since, as can be seen,the gold dots surround and dot the proteosome vesicles, the image ofproteosome vaccines with LPS non-covalently linked to the proteosomes isthereby confirmed and visualized. Surprisingly, the consistent vesicularnature of the vaccines made with the HF technology of the instantinvention was not found when the DT preparations using the oldertechnology were examined by electron microscopy (unpublished results).

5.12 Proteosome-P. Shigelloides LPS Vaccine Protects Against LethalShigella Sonnei Pneumonia in an Animal Model of Shigellosis

As can be seen in FIG. 5, the proteosome-P. shigelloides LPS vaccineconsistently confers highly significant protection against lethalinfection with P. shigelloides as measured in an animal model ofshigellosis in which the animals are challenged with live organisms toinduce lethal pneumonia. These data also demonstrate that not only arethe correct and protective antibodies produced by the proteosome-P.shigelloides LPS vaccine, but also that this intranasal sub-unit vaccinecan protect against lethal pneumonia. Groups of outbred mice (14 to 15per group) were immunized intranasally on weeks zero and three with theproteosome-P. shigelloides LPS vaccine (10 micrograms each ofproteosomes and LPS per 20 mircoliters). Control groups were givensaline on the same schedule. All mice were challenged intranasally withfive to ten million colony forming units of S. sonnei on week seven. Themice were observed daily for two weeks after challenge, and mortalityresulting from the ensuing pulmonary disease was recorded. As we haveshown previously, 86 to 100% of the control mice die within four daysafter challenge. In both experiments, 93% of mice immunized with thevaccine survived challenge (P<0.001, Fisher's Exact Test).

5.13 Intranasal or Oral Immunization with Proteosome-Shigella flexneri2a LPS Vaccines Induce Anti-Shigella LPS IgG and IgA in Serum and IgA inIntestinal and Lung Lavage Fluids That Can Last 30-60 DaysPost-immunization.

As shown in FIG. 6, two immunizations with proteosome-shigella flexneri2a LPS vaccines induce antibodies in sera and in lung and intestinalsecretions that can last 30 to 60 days post immunization. These datashow that proteosome vaccines can stimulate the common mucosal immunesystem to secrete specific antibodies even at locations far away fromthe immunizing site. Specifically, intranasal immunization can induceantibodies in intestinal secretions and vice versa. This ability expandsthe potential utility of proteosome vaccines to protect againstpathogens that invade the host through the mucosal portals of entrythroughout the body.

5.14 FIG. 7 shows the induction of anti-shigella LPS IgA, IgG and IgMperipheral blood ASC responses, and serum, salivary and urinary IgA andIgG antibody responses after intranasal or oral immunization of humanvolunteers with one or two doses proteosome-P. shigelloides LPS vaccinesfor Shigella sonnei using different vaccine amounts. As shown, intraasalimmunization elicited responses in a dose dependant fashion with thehighest dose inducing responses in all six volunteers.

IgA, IgG and IgM serum responses were elicited in each of the intranasalgroups. In addition, strong antibody secreting cell ASC responses wereinduced indicating that mucosal immunization stimulated trafficking ofantibody secreting cells—most notably, IgA-secreting cells. Mostimportantly, IgA responses were also found in salivary and, especially,urinary samples indicating that secretory IgA was produced at mucosalsurfaces (since IgA does not pass through the kidney, the urinary IgAreflects local secretion of antibody and suggests that such intranasalvaccines also produce local intestinal IgA and that intranasal vaccinescould be used to protect against urinary tract infections caused by gramnegative organisms.) It is noteworthy that the majority of these ASC andantibody responses were found after only one immunization suggestingthat booster immunization may not be necessary. IgA and IgG ASCs andurinary IgA were also found after oral immunization. The most effectiveuse of oral administration may be as a booster immunization after nasalpriming, if necessary.

5.15 Demonstration of non-covalent complexing of the multimolecularproteosome proteins and LPS is shown in FIG. 8. The graph shows LPS inHPLC fractions after applying uncomplexed P. shigelloides LPS whichoccurs at a different part of the column elution profile than whenapplying the proteosome-LPS vaccine. The co-elution of LPS and proteinsin HPLC fractions after applying proteosome-P. shigelloides LPS vaccineis shown with peaks that correspond to the largest protein aggregates ofproteosomes. The data were measured using an inhibition ELISA toquantitate the LPS and using the OD at A280 to quantitate the proteosomeproteins. The HPLC column was a Tosohaas G50000Pwxl and the molecularweight standards are indicated by the arrows.

What is claimed is:
 1. A continuous process for the preparation of proteosome-amphiphilic determinant vaccine comprising a) forming a mixture of proteosome, a detergent and an amphiphilic determinant, b) continuously subjecting the mixture to a diafiltration or an ultrafiltration system to remove the detergent and form an amphiphilic determinant-proteosome complex in the retentate under non-static conditions, c) monitoring the amount of amphiphilic determinant-proteosome complex formation, and d) recovering the amphiphilic determinant-proteosome complex, wherein the nominal molecular weight cutoff of the system is about 3,000 or greater.
 2. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 wherein the ultrafiltration involves tangential flow.
 3. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 wherein the ultrafiltration system is selected from a hollow fiber cartridge, platform membrane and membrane cartridge.
 4. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 further comprising monitoring of the detergent by continual measuring of permeate samples.
 5. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 wherein monitoring includes measuring the optical density of permeate or retentate sample.
 6. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 wherein the monitoring involves measuring the amount of detergent removal by mixing a permeate sample with a known amount of SDS (sodium dodecyl sulfate) to form a precipitate if detergent is present, and determining the amount of precipitation.
 7. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 6 wherein the proteosome-amphiphilic determinant complex is recovered when the detergent in the permeate has been essentially removed.
 8. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 wherein the detergent is Empigen BB.
 9. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 wherein the amphiphilic determinant is a lipopolysaccharide.
 10. A process for the preparation of proteosome-lipopolysaccharide vaccine according to claim 9 wherein the lipopolysaccharide is obtained from a gram negative bacteria.
 11. A process for the preparation of proteosome-lipopolysaccharide vaccine according to claim 10 wherein the gram negative bacteria is a plesiomonas.
 12. A process for the preparation of proteosome-lipopolysaccharide vaccine according to claim 11 wherein the plesiomonas is Plesiomonas shigelloides.
 13. A process for the preparation of proteosome-lipopolysaccharide vaccine according to claim 10 wherein the gram negative bacteria is a shigella.
 14. A process for the preparation of proteosome-lipopolysaccharide vaccine according to claim 13 wherein the shigella is Shigella flexneri, Shigella sonnei, or Shigella boydii.
 15. A process for the preparation of proteosome-lipopolysaccharide vaccine according to claim 10 wherein the gram negative bacteria is a neisseria.
 16. A process for the preparation of proteosome-lipopolysaccharide vaccine according to claim 15 wherein the neisseia is Neisseria meningitidis.
 17. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 wherein the proteosome is derived from N. meningiditis or N. gonorrhea.
 18. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 wherein the recovery of the LPS-proteosome complex is effected when the monitored rate of detergent removal is decreasing or constant.
 19. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 1 further comprising e) mixing the recovered proteosome-amphiphilic determinant complex with a physiologically acceptable carrier to form a vaccine.
 20. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 19 further comprising concentrating the proteosome-amphiphilic determinant complex prior to its recovery.
 21. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 20 wherein the concentration is continued to a desired final concentration.
 22. A process for the preparation of proteosome-amphiphilic determinant vaccine according to claim 19 wherein the process is performed with different amphiphilic determinants types to form a series of proteosome-amphiphilic determinant complexes that are assembled in step e) to form a multivalent vaccine.
 23. A multivalent vaccine prepared by the process of claim
 22. 24. A process for protecting against a disease comprising administering the vaccine of claim 23 in an effective amount to a recipient.
 25. A process for protecting against a disease comprising administering the vaccine prepared according to the process of claim 23 to a recipient. 